Isolation of peripheral blood CD4(+) T cells using RosetteSep and MACS for studies of DNA turnover by deuterium labeling.

Tracking deuterium ((2)H) incorporation into cellular DNA, after administration of (2)H(2)O or (2)H(2)-glucose, is a recently developed, broadly applicable method for measuring in vivo cell proliferation and turnover that can be used safely in humans. This approach has been used to evaluate the turnover of T-cell subpopulations purified from the peripheral blood of HIV-1-infected patients using fluorescence-activated cell sorting (FACS). A requirement for widespread adoption of this approach for medical decision-making and for use in larger clinical trials is a simple, reproducible, high-throughput method for isolation of highly purified CD4(+) T cells from peripheral blood. Here, we present a simple method, which does not require FACS, for isolating these cells in sufficient purity and yield for analysis of (2)H incorporation into DNA. When blood from HIV-1-infected patients was used, neither the depletion of unwanted cell lineages by erythrocyte crosslinking (RosetteSep) nor the enrichment of CD4(+) cells by immunomagnetic beads (MACS) individually resulted in sufficient purity. The successive application of the two techniques, however, permitted isolation of >95% pure CD4(+) T cells in adequate yield (>10(6) cells/10 ml blood) from healthy donors and HIV-1-infected patients with CD4 counts between 300 and 700 cells/microl. Moreover, (2)H incorporation into cellular DNA after administration of (2)H(2)O to HIV-1-infected patients was indistinguishable between CD4(+) T cells isolated by RosetteSep/MACS and FACS. Thus, both FACS and the new method isolate a similar mixture of long- and short-lived CD4(+) T cells. In practice, the RosetteSep/MACS method is simple, rapid, robust and capable of high throughput.

Development and validation of an assay for measuring preimplantation factor (PIF) of embryonal origin.

PROBLEM: Tests to determine presence of embryos prior to implantation are needed. METHODS: Sera from women after embryo transfer were tested for preimplantation factor (PIF) using the lymphocyte/platelet binding assay. Autorosettes were counted using blood type O+ donor lymphocytes and platelets incubated with blinded serum in the presence of antiCD2 antibody and rabbit complement. Human chorion gonadotropin (hCG) concentrations were determined 7 days later and compared with results of the lymphocyte/platelet assay. Implantation was confirmed by ultrasonographic evidence of presence of an intrauterine gestational sac. The roles of platelet activating factor (PAF) and chaperonin 10 in the observed phenomena were studied experimentally. RESULTS: Significantly more lymphocyte/platelet rosette formations were observed when sera from women who successfully implanted were compared to sera from women who failed to implant. Neither PAF nor chaparonin added to the tested sera controls influenced the percentage of lymphocyte/platelets rosettes. CONCLUSIONS: PIF is a likely candidate to be the next frontier of diagnosing the presence of viable preimplantation embryos in vivo.